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Georg Fuchs - One of the best experts on this subject based on the ideXlab platform.
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structural basis for a bispecific nadp and coa binding site in an archaeal malonyl coenzyme a reductase
Journal of Biological Chemistry, 2013Co-Authors: Ulrike Demmer, Georg Fuchs, Daniel Kockelkorn, Eberhard Warkentin, Ankita Srivastava, Markus Potter, Achim Marx, Ulrich ErmlerAbstract:Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of Succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP+ and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP+ and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3′- and 2′-ribose phosphate group of CoA and NADP+, respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP+. Evolution from an NADP+ to a bispecific NADP+ and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP+ binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis. Background: Malonyl-CoA reductase of the CO2 assimilating 3-hydroxypropionate/4-hydroxybutyrate cycle reduces malonyl-CoA to malonic semialdehyde. Results: Malonyl-CoA reductase complexed with CoA and NADP+ was structurally characterized. Conclusion: The protein acts as rigid template to press CoA and NADP+ into similar S-shaped, superimposable forms. Significance: The data indicate how to construct a bispecific cofactor binding site and to engineer a malonyl- into methyl-malonyl-CoA reductase for polyester building block production.
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Identification of missing genes and enzymes for autotrophic carbon fixation in crenarchaeota.
Journal of bacteriology, 2010Co-Authors: W. H. Ramos-vera, Daniel Kockelkorn, M. Weiss, Eric Strittmatter, Georg FuchsAbstract:Two autotrophic carbon fixation cycles have been identified in Crenarchaeota. The dicarboxylate/4-hydroxybutyrate cycle functions in anaerobic or microaerobic autotrophic members of the Thermoproteales and Desulfurococcales. The 3-hydroxypropionate/4-hydroxybutyrate cycle occurs in aerobic autotrophic Sulfolobales; a similar cycle may operate in autotrophic aerobic marine Crenarchaeota. Both cycles form succinyl-coenzyme A (CoA) from acetyl-CoA and two molecules of inorganic carbon, but they use different means. Both cycles have in common the (re)generation of acetyl-CoA from Succinyl-CoA via identical intermediates. Here, we identified several missing enzymes/genes involved in the seven-step conversion of Succinyl-CoA to two molecules of acetyl-CoA in Thermoproteus neutrophilus (Thermoproteales), Ignicoccus hospitalis (Desulfurococcales), and Metallosphaera sedula (Sulfolobales). The identified enzymes/genes include Succinyl-CoA reductase, succinic semialdehyde reductase, 4-hydroxybutyrate-CoA ligase, bifunctional crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase, and beta-ketothiolase. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes a mechanistically intriguing elimination of water, is well conserved and rightly can be considered the key enzyme of these two cycles. In contrast, several of the other enzymes evolved from quite different sources, making functional predictions based solely on genome interpretation difficult, if not questionable.
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malonic semialdehyde reductase succinic semialdehyde reductase and succinyl coenzyme a reductase from metallosphaera sedula enzymes of the autotrophic 3 hydroxypropionate 4 hydroxybutyrate cycle in sulfolobales
Journal of Bacteriology, 2009Co-Authors: Daniel Kockelkorn, Georg FuchsAbstract:A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO2 fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and Succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to Succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/Succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation.
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a dicarboxylate 4 hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic archaeum ignicoccus hospitalis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Martin Gallenberger, Ivan A. Berg, Ulrike Jahn, Eva Eylert, Daniel Kockelkorn, Wolfgang Eisenreich, Georg FuchsAbstract:Ignicoccus hospitalis is an anaerobic, autotrophic, hyperthermophilic Archaeum that serves as a host for the symbiotic/parasitic Archaeum Nanoarchaeum equitans. It uses a yet unsolved autotrophic CO2 fixation pathway that starts from acetyl-CoA (CoA), which is reductively carboxylated to pyruvate. Pyruvate is converted to phosphoenol-pyruvate (PEP), from which glucogenesis as well as oxaloacetate formation branch off. Here, we present the complete metabolic cycle by which the primary CO2 acceptor molecule acetyl-CoA is regenerated. Oxaloacetate is reduced to Succinyl-CoA by an incomplete reductive citric acid cycle lacking 2-oxoglutarate dehydrogenase or synthase. Succinyl-CoA is reduced to 4-hydroxybutyrate, which is then activated to the CoA thioester. By using the radical enzyme 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoA is dehydrated to crotonyl-CoA. Finally, β-oxidation of crotonyl-CoA leads to two molecules of acetyl-CoA. Thus, the cyclic pathway forms an extra molecule of acetyl-CoA, with pyruvate synthase and PEP carboxylase as the carboxylating enzymes. The proposal is based on in vitro transformation of 4-hydroxybutyrate, detection of all enzyme activities, and in vivo-labeling experiments using [1-14C]4-hydroxybutyrate, [1,4-13C2], [U-13C4]succinate, or [1-13C]pyruvate as tracers. The pathway is termed the dicarboxylate/4-hydroxybutyrate cycle. It combines anaerobic metabolic modules to a straightforward and efficient CO2 fixation mechanism.
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Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.
Journal of bacteriology, 2006Co-Authors: Silke Friedmann, Birgit E Alber, Georg FuchsAbstract:The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the Succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of Succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for Succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + Succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for Succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.
Marie E Fraser - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for the binding of succinate to Succinyl-CoA synthetase.
Acta Crystallographica Section D Structural Biology, 2016Co-Authors: Ji Huang, Marie E FraserAbstract:Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific Succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 A resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why Succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with l-malate.
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Structure of GTP-specific Succinyl-CoA synthetase in complex with CoA.
Acta Crystallographica Section F Structural Biology Communications, 2015Co-Authors: Ji Huang, Manpreet Malhi, Jan Deneke, Marie E FraserAbstract:Pig GTP-specific Succinyl-CoA synthetase is an αβ-heterodimer. The crystal structure of the complex with the substrate CoA was determined at 2.1 A resolution. The structure shows CoA bound to the amino-terminal domain of the α-subunit, with the free thiol extending from the adenine portion into the site where the catalytic histidine residue resides.
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Truncated Human ATP-Specific Succinyl-CoA Synthetase
Acta Crystallographica Section A Foundations and Advances, 2014Co-Authors: Marie E Fraser, Koto HayakawaAbstract:Two isoforms of the heterodimeric enzyme Succinyl-CoA synthetase (SCS) exist in the mitochondria of humans. One is specific for ATP, while the other is specific for GTP. Both catalyze the reversible reaction: succinate + CoA + NTP ⇌ Succinyl-CoA + NDP + Pi, where N denotes adenosine or guanosine. SCS is best known as an enzyme of the citric acid cycle where the reaction generates NTP. In the reverse direction, SCS replenishes Succinyl-CoA required for the catabolism of ketone bodies and for heme synthesis. Nucleotide-specific forms are thought to be required for SCS to serve its different metabolic roles. The nucleotide specificity lies in the β-subunit [1], and the β-subunit of human ATP-specific SCS has been shown to interact with the C-terminus of erythroid-specific aminolevulinic acid synthase (ALAS2) [2]. ALAS2 catalyzes the committed step in heme synthesis: Succinyl-CoA + Gly ⇌ 5-aminolevulinate + CoA + CO2. An interaction between SCS and ALAS2 makes biological sense, since this could provide channeling of Succinyl-CoA from SCS to ALAS2. We hypothesize that the interaction is with the carboxy-terminus of the β-subunit of ATP-specific SCS because sequence comparisons show that the β-subunit of ATP-specific SCS has a carboxy-terminal extension when compared to other SCSs' β-subunits. To test this hypothesis, we added a carboxy-terminal His8-tag to the α-subunit of human ATP-specific SCS and mutated the codon for Thr 396β to a stop codon. This truncated version of human ATP-specific SCS has been produced in E. coli and purified. As well as testing to see if truncated human ATP-specific SCS interacts with ALAS2, we are using the truncated version in crystallization trials. Crystals of full-length human ATP-specific SCS diffract to only 3.2 Å and our goal is to obtain better-diffracting crystals of the complex of ATP with truncated human ATP-specific SCS.
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X-linked sideroblastic anemia due to carboxyl-terminal ALAS2 mutations that cause loss of binding to the β-subunit of Succinyl-CoA synthetase (SUCLA2).
The Journal of biological chemistry, 2012Co-Authors: David F. Bishop, Marie E Fraser, Vassili Tchaikovskii, A. Victor Hoffbrand, Steven MargolisAbstract:Mutations in the erythroid-specific aminolevulinic acid synthase gene (ALAS2) cause X-linked sideroblastic anemia (XLSA) by reducing mitochondrial enzymatic activity. Surprisingly, a patient with the classic XLSA phenotype had a novel exon 11 mutation encoding a recombinant enzyme (p.Met567Val) with normal activity, kinetics, and stability. Similarly, both an expressed adjacent XLSA mutation, p.Ser568Gly, and a mutation (p.Phe557Ter) lacking the 31 carboxyl-terminal residues also had normal or enhanced activity, kinetics, and stability. Because ALAS2 binds to the β subunit of Succinyl-CoA synthetase (SUCLA2), the mutant proteins were tested for their ability to bind to this protein. Wild type ALAS2 bound strongly to a SUCLA2 affinity column, but the adjacent XLSA mutant enzymes and the truncated mutant did not bind. In contrast, vitamin B6-responsive XLSA mutations p.Arg452Cys and p.Arg452His, with normal in vitro enzyme activity and stability, did not interfere with binding to SUCLA2 but instead had loss of positive cooperativity for Succinyl-CoA binding, an increased K(m) for Succinyl-CoA, and reduced vitamin B6 affinity. Consistent with the association of SUCLA2 binding with in vivo ALAS2 activity, the p.Met567GlufsX2 mutant protein that causes X-linked protoporphyria bound strongly to SUCLA2, highlighting the probable role of an ALAS2-Succinyl-CoA synthetase complex in the regulation of erythroid heme biosynthesis.
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Participation of Cys123α of Escherichia coli Succinyl-CoA synthetase in catalysis
Acta Crystallographica Section D Biological Crystallography, 2007Co-Authors: Esther Hidber, Edward R. Brownie, Koto Hayakawa, Marie E FraserAbstract:Succinyl-CoA synthetase has a highly conserved cysteine residue, Cys123α in the Escherichia coli enzyme, that is located near the CoA-binding site and the active-site histidine residue. To test whether the succinyl moiety of Succinyl-CoA is transferred to the thiol of Cys123α as part of the catalytic mechanism, this residue was mutated to alanine, serine, threonine and valine. Each mutant protein was catalytically active, although less active than the wild type. This proved that the specific formation of a thioester bond with Cys123α is not part of the catalytic mechanism. To understand why the mutations affected catalysis, the crystal structures of the four mutant proteins were determined. The alanine mutant showed no structural changes yet had reduced activity, suggesting that the size of the cysteine is important for optimal activity. These results explain why this cysteine residue is conserved in the sequences of Succinyl-CoA synthetases from different sources.
Daniel Kockelkorn - One of the best experts on this subject based on the ideXlab platform.
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structural basis for a bispecific nadp and coa binding site in an archaeal malonyl coenzyme a reductase
Journal of Biological Chemistry, 2013Co-Authors: Ulrike Demmer, Georg Fuchs, Daniel Kockelkorn, Eberhard Warkentin, Ankita Srivastava, Markus Potter, Achim Marx, Ulrich ErmlerAbstract:Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of Succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP+ and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP+ and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3′- and 2′-ribose phosphate group of CoA and NADP+, respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP+. Evolution from an NADP+ to a bispecific NADP+ and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP+ binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis. Background: Malonyl-CoA reductase of the CO2 assimilating 3-hydroxypropionate/4-hydroxybutyrate cycle reduces malonyl-CoA to malonic semialdehyde. Results: Malonyl-CoA reductase complexed with CoA and NADP+ was structurally characterized. Conclusion: The protein acts as rigid template to press CoA and NADP+ into similar S-shaped, superimposable forms. Significance: The data indicate how to construct a bispecific cofactor binding site and to engineer a malonyl- into methyl-malonyl-CoA reductase for polyester building block production.
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Identification of missing genes and enzymes for autotrophic carbon fixation in crenarchaeota.
Journal of bacteriology, 2010Co-Authors: W. H. Ramos-vera, Daniel Kockelkorn, M. Weiss, Eric Strittmatter, Georg FuchsAbstract:Two autotrophic carbon fixation cycles have been identified in Crenarchaeota. The dicarboxylate/4-hydroxybutyrate cycle functions in anaerobic or microaerobic autotrophic members of the Thermoproteales and Desulfurococcales. The 3-hydroxypropionate/4-hydroxybutyrate cycle occurs in aerobic autotrophic Sulfolobales; a similar cycle may operate in autotrophic aerobic marine Crenarchaeota. Both cycles form succinyl-coenzyme A (CoA) from acetyl-CoA and two molecules of inorganic carbon, but they use different means. Both cycles have in common the (re)generation of acetyl-CoA from Succinyl-CoA via identical intermediates. Here, we identified several missing enzymes/genes involved in the seven-step conversion of Succinyl-CoA to two molecules of acetyl-CoA in Thermoproteus neutrophilus (Thermoproteales), Ignicoccus hospitalis (Desulfurococcales), and Metallosphaera sedula (Sulfolobales). The identified enzymes/genes include Succinyl-CoA reductase, succinic semialdehyde reductase, 4-hydroxybutyrate-CoA ligase, bifunctional crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase, and beta-ketothiolase. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes a mechanistically intriguing elimination of water, is well conserved and rightly can be considered the key enzyme of these two cycles. In contrast, several of the other enzymes evolved from quite different sources, making functional predictions based solely on genome interpretation difficult, if not questionable.
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malonic semialdehyde reductase succinic semialdehyde reductase and succinyl coenzyme a reductase from metallosphaera sedula enzymes of the autotrophic 3 hydroxypropionate 4 hydroxybutyrate cycle in sulfolobales
Journal of Bacteriology, 2009Co-Authors: Daniel Kockelkorn, Georg FuchsAbstract:A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO2 fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and Succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to Succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/Succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation.
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a dicarboxylate 4 hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic archaeum ignicoccus hospitalis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Martin Gallenberger, Ivan A. Berg, Ulrike Jahn, Eva Eylert, Daniel Kockelkorn, Wolfgang Eisenreich, Georg FuchsAbstract:Ignicoccus hospitalis is an anaerobic, autotrophic, hyperthermophilic Archaeum that serves as a host for the symbiotic/parasitic Archaeum Nanoarchaeum equitans. It uses a yet unsolved autotrophic CO2 fixation pathway that starts from acetyl-CoA (CoA), which is reductively carboxylated to pyruvate. Pyruvate is converted to phosphoenol-pyruvate (PEP), from which glucogenesis as well as oxaloacetate formation branch off. Here, we present the complete metabolic cycle by which the primary CO2 acceptor molecule acetyl-CoA is regenerated. Oxaloacetate is reduced to Succinyl-CoA by an incomplete reductive citric acid cycle lacking 2-oxoglutarate dehydrogenase or synthase. Succinyl-CoA is reduced to 4-hydroxybutyrate, which is then activated to the CoA thioester. By using the radical enzyme 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoA is dehydrated to crotonyl-CoA. Finally, β-oxidation of crotonyl-CoA leads to two molecules of acetyl-CoA. Thus, the cyclic pathway forms an extra molecule of acetyl-CoA, with pyruvate synthase and PEP carboxylase as the carboxylating enzymes. The proposal is based on in vitro transformation of 4-hydroxybutyrate, detection of all enzyme activities, and in vivo-labeling experiments using [1-14C]4-hydroxybutyrate, [1,4-13C2], [U-13C4]succinate, or [1-13C]pyruvate as tracers. The pathway is termed the dicarboxylate/4-hydroxybutyrate cycle. It combines anaerobic metabolic modules to a straightforward and efficient CO2 fixation mechanism.
Edward R. Brownie - One of the best experts on this subject based on the ideXlab platform.
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Participation of Cys123alpha of Escherichia coli Succinyl-CoA synthetase in catalysis.
Acta crystallographica. Section D Biological crystallography, 2007Co-Authors: Esther Hidber, Edward R. Brownie, Koto Hayakawa, Marie E FraserAbstract:Succinyl-CoA synthetase has a highly conserved cysteine residue, Cys123alpha in the Escherichia coli enzyme, that is located near the CoA-binding site and the active-site histidine residue. To test whether the succinyl moiety of Succinyl-CoA is transferred to the thiol of Cys123alpha as part of the catalytic mechanism, this residue was mutated to alanine, serine, threonine and valine. Each mutant protein was catalytically active, although less active than the wild type. This proved that the specific formation of a thioester bond with Cys123alpha is not part of the catalytic mechanism. To understand why the mutations affected catalysis, the crystal structures of the four mutant proteins were determined. The alanine mutant showed no structural changes yet had reduced activity, suggesting that the size of the cysteine is important for optimal activity. These results explain why this cysteine residue is conserved in the sequences of Succinyl-CoA synthetases from different sources.
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Participation of Cys123α of Escherichia coli Succinyl-CoA synthetase in catalysis
Acta Crystallographica Section D Biological Crystallography, 2007Co-Authors: Esther Hidber, Edward R. Brownie, Koto Hayakawa, Marie E FraserAbstract:Succinyl-CoA synthetase has a highly conserved cysteine residue, Cys123α in the Escherichia coli enzyme, that is located near the CoA-binding site and the active-site histidine residue. To test whether the succinyl moiety of Succinyl-CoA is transferred to the thiol of Cys123α as part of the catalytic mechanism, this residue was mutated to alanine, serine, threonine and valine. Each mutant protein was catalytically active, although less active than the wild type. This proved that the specific formation of a thioester bond with Cys123α is not part of the catalytic mechanism. To understand why the mutations affected catalysis, the crystal structures of the four mutant proteins were determined. The alanine mutant showed no structural changes yet had reduced activity, suggesting that the size of the cysteine is important for optimal activity. These results explain why this cysteine residue is conserved in the sequences of Succinyl-CoA synthetases from different sources.
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Interactions of GTP with the ATP-grasp domain of GTP-specific Succinyl-CoA synthetase.
The Journal of biological chemistry, 2006Co-Authors: Marie E Fraser, Koto Hayakawa, Millicent S. Hume, David G. Ryan, Edward R. BrownieAbstract:Two isoforms of Succinyl-CoA synthetase exist in mammals, one specific for ATP and the other for GTP. The GTP-specific form of pig Succinyl-CoA synthetase has been crystallized in the presence of GTP and the structure determined to 2.1 A resolution. GTP is bound in the ATP-grasp domain, where interactions of the guanine base with a glutamine residue (Gln-20β) and with backbone atoms provide the specificity. The γ-phosphate interacts with the side chain of an arginine residue (Arg-54β) and with backbone amide nitrogen atoms, leading to tight interactions between the γ-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the γ-phosphate is exposed, free to react with the other substrate. To test if GDP would interact with GTP-specific Succinyl-CoA synthetase in the same way that ADP interacts with other members of the family of ATP-grasp proteins, the structure of GDP bound to GTP-specific Succinyl-CoA synthetase was also determined. A comparison of the conformations of GTP and GDP shows that the bases adopt the same position but that changes in conformation of the ribose moieties and the α- and β-phosphates allow the γ-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the β-phosphate interacts with these residues in GDP. The complex of GTP with Succinyl-CoA synthetase shows that the enzyme is able to protect GTP from hydrolysis when the active-site histidine residue is not in position to be phosphorylated.
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Probing the Nucleotide-Binding Site of Escherichia coli Succinyl-CoA Synthetase†
Biochemistry, 1999Co-Authors: Michael A. Joyce, Edward R. Brownie, William A. Bridger, Marie E Fraser, Michael N G James, William T. WolodkoAbstract:Succinyl-CoA synthetase (SCS) catalyzes the reversible interchange of purine nucleoside diphosphate, Succinyl-CoA, and Pi with purine nucleoside triphosphate, succinate, and CoA via a phosphorylated histidine (H246α) intermediate. Two potential nucleotide-binding sites were predicted in the β-subunit, and have been differentiated by photoaffinity labeling with 8-N3-ATP and by site-directed mutagenesis. It was demonstrated that 8-N3-ATP is a suitable analogue for probing the nucleotide-binding site of SCS. Two tryptic peptides from the N-terminal domain of the β-subunit were labeled with 8-N3-ATP. These corresponded to residues 107−119β and 121−146β, two regions lying along one side of an ATP-grasp fold. A mutant protein with changes on the opposite side of the fold (G53βV/R54βE) was unable to be phosphorylated using ATP or GTP, but could be phosphorylated by Succinyl-CoA and Pi. A mutant protein designed to probe nucleotide specificity (P20βQ) had a Km(app) for GTP that was more than 5 times lower than th...
Johann Heider - One of the best experts on this subject based on the ideXlab platform.
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Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase: an Enzyme of the Anaerobic Toluene Catabolic Pathway in Denitrifying Bacteria
Journal of Bacteriology, 2001Co-Authors: Christina Leutwein, Johann HeiderAbstract:Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (R)-benzylsuccinate as first intermediate, which is further metabolized via β-oxidation to benzoyl-coenzyme A (CoA) and Succinyl-CoA. A specific Succinyl-CoA:(R)-benzylsuccinate CoA-transferase activating (R)-benzylsuccinate to the CoA-thioester was purified and characterized from Thauera aromatica. The enzyme is fully reversible and forms exclusively the 2-(R)-benzylSuccinyl-CoA isomer. Only some close chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (R)-benzylsuccinate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate. In contrast to all other known CoA-transferases, the enzyme consists of two subunits of similar amino acid sequences and similar sizes (44 and 45 kDa) in an α2β2 conformation. Identity of the subunits with the products of the previously identified toluene-induced bbsEF genes was confirmed by determination of the exact masses via electrospray-mass spectrometry. The deduced amino acid sequences resemble those of only two other characterized CoA-transferases, oxalyl-CoA:formate CoA-transferase and (E)-cinnamoyl-CoA:(R)-phenyllactate CoA-transferase, which represent a new family of CoA-transferases. As suggested by kinetic analysis, the reaction mechanism of enzymes of this family apparently involves formation of a ternary complex between the enzyme and the two substrates.
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Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and β-oxidation of the first intermediate, (R)-(+)-benzylsuccinate
Microbiology, 1999Co-Authors: Christina Leutwein, Johann HeiderAbstract:Anaerobic catabolism of toluene is initiated by addition of the methyl group of toluene to the double bond of a fumarate cosubstrate to yield the first intermediate, benzylsuccinate. This reaction is catalysed by the glycyl-radical enzyme benzylsuccinate synthase, as shown for the denitrifying bacterium Thauera aromatica. Benzylsuccinate is further oxidized to benzoyl-CoA, the central intermediate of anaerobic degradation of aromatic compounds. The authors show here by experiments with cell extracts of toluene-grown T. aromatica that the pathway of benzylsuccinate oxidation requires activation of the free acid to a CoA-thioester, catalysed by a toluene-induced, reversible Succinyl-CoA-dependent CoA-transferase. The product of the CoA-transferase reaction, benzylSuccinyl-CoA, is oxidized to benzoyl-CoA and Succinyl-CoA in extracts of toluene-grown cells, adding proof to the proposed anaerobic toluene-catabolic pathway. The stereochemical preferences of the enzymes catalysing formation and activation of benzylsuccinate have been analysed. Benzylsuccinate synthase was found to produce exclusively (R)-(+)-benzylsuccinate, although the proposed reaction mechanism of this enzyme proceeds via radical intermediates. In accordance, the reaction of Succinyl-CoA:benzylsuccinate CoA-transferase is also specific for (R)-(+)-benzylsuccinate and does not proceed with the (S)-(−)-enantiomer.
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Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and beta-oxidation of the first intermediate, (R)-(+)-benzylsuccinate.
Microbiology (Reading England), 1999Co-Authors: Christina Leutwein, Johann HeiderAbstract:Anaerobic catabolism of toluene is initiated by addition of the methyl group of toluene to the double bond of a fumarate cosubstrate to yield the first intermediate, benzylsuccinate. This reaction is catalysed by the glycyl-radical enzyme benzylsuccinate synthase, as shown for the denitrifying bacterium Thauera aromatica. Benzylsuccinate is further oxidized to benzoyl-CoA, the central intermediate of anaerobic degradation of aromatic compounds. The authors show here by experiments with cell extracts of toluene-grown T. aromatica that the pathway of benzylsuccinate oxidation requires activation of the free acid to a CoA-thioester, catalysed by a toluene-induced, reversible Succinyl-CoA-dependent CoA-transferase. The product of the CoA-transferase reaction, benzylSuccinyl-CoA, is oxidized to benzoyl-CoA and Succinyl-CoA in extracts of toluene-grown cells, adding proof to the proposed anaerobic toluene-catabolic pathway. The stereochemical preferences of the enzymes catalysing formation and activation of benzylsuccinate have been analysed. Benzylsuccinate synthase was found to produce exclusively (R)-(+)-benzylsuccinate, although the proposed reaction mechanism of this enzyme proceeds via radical intermediates. In accordance, the reaction of Succinyl-CoA:benzylsuccinate CoA-transferase is also specific for (R)-(+)-benzylsuccinate and does not proceed with the (S)-(-)-enantiomer.
